Project Summary - Project 3 This Program Project Grant aims to define the sites and mechanisms of general anesthetics on pentameric ligand-gated ion channels, specifically heteromeric GABAA and glycine receptors, and to develop novel compounds with greater selectivity for the various unique sites on these receptors. Our photolabeling and structure-function studies have established a novel and cohesive paradigm for the molecular mechanism of etomidate at its major targets, a subset of GABAA receptors. Etomidate binds in the extracellular portion of two of the five transmembrane subunit interfaces in heteromeric abg GABAARs, and functions as an allosteric co- agonist. Preliminary data indicate that other potent anesthetics that target GABAARs, including novel barbiturates (mTFD-MPAB) and propofol derivatives, act variously at the etomidate sites and/or homologs of these sites in other subunit interfaces, with different degrees of specificity. In contrast, data suggest that alphaxalone binds deeper within the transmembrane subunit interfaces. We further hypothesize that several types of subunit interfacial anesthetic sites are formed in heteromeric GlyRs, and that these are also homologs of sites in GABAA receptors. A corollary of our hypothesis is that differential anesthetic sensitivity of different GABAAR subtypes is due to natural sequence variations in the anesthetic binding sites. The aims of Project 3 are to: 1) assess the functional roles of amino acids in the different unique anesthetic sites of several key abg and abd GABAARs as well as a1b GlyRs; 2) to develop quantitative models accounting for the effects of etomidate, propofol, mTFD-MPAB, and alphaxalone at each unique interfacial site and in different types of receptors; 3) identify mutations within each site that selectively ablate anesthetic effects; and 4) test novel compounds for modulatory potency and efficacy in both GABAA and Gly receptors. Based on photolabeling data from Project 1 and homology models of the transmembrane subunit interfacial cavities (Protein Chemistry Core; C), Project 3 (supported by the Protein Synthesis Core; D) will mutate single residues to tryptophan (hypothesized to mimic bound anesthetic) and electrophysiologically assess mutant effects on receptor gating, GABA sensitivity, and anesthetic sensitivity. We will quantify mutant effects using the formal mechanistic framework of two-state Monod-Wyman-Changeux allosteric co-agonism, and apply MWC models with unique binding and efficacy parameters at distinct anesthetic sites to test whether these sites equally and independently contribute to anesthetic effects. Mutations that selectively ablate anesthetic actions in single sites will be identified for drug screening in this Project and Project 2. We will also help screen novel compounds synthesized by the Synthetic Chemistry Core (B). Thus, this project synergizes with the other projects and scientific cores to help achieve the overall aims of the P01.